The overall objective of this proposed research is to establish the mechanisms responsible for the short-term effects of glucagon, insulin, and epinephrine on hepatic glycolysis, gluconeogenesis, and lipogenesis. The studies will be conducted with isolated hepatocytes prepared from meal-fed, fasted, and diabetic rats. The specific aims include: (a) to determine whether interconversion of phosphofructokinase into an inactive form is involved in glucagon and epinephrine inhibition of hepatic glycolysis and stimulation of gluconeogenesis; (b) to further investigate the significance of hormone-mediated interconversion of pyruvate kinase to the regulation of hepatic aerobic glycolysis, gluconeogenesis, and lipogenesis; and (c) to determine the mechanism responsible for glucagon inhibition of hepatic lipogenesis. Studies from this laboratory have shown that the inhibition of hepatic lipogenesis by glucagon is explained in part by the inhibitory effects of cyclic AMP upon aerobic glycolysis. However, neither the mechanism responsible for cyclic AMP inhibition of aerobic glycolysis nor the basis for the requirement of a rather high concentration of lactate plus pyruvate for lipogenesis has been firmly established. Our working hypothesis is that phosphofructokinase and pyruvate kinase are the most important regulatory enzymes involved in short-term control of aerobic glycolysis and gluconeogenesis by glucagon and insulin. The requirement for high concentrations of lactate plus pyruvate for rapid rates of hepatic lipogenesis may be explained by pyruvate activation of pyruvate dehydrogenase along with an accumulation of cytoplasmic citrate which may activate acetyl-CoA carboxylase. One of the major goals of the proposed research is to determine whether the steady state concentration of cytoplasmic citrate is a primary determinant of the rate of de novo fatty acid synthesis by the liver.